Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

ABSTRACT

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

STATEMENT REGARDING FEDERAL RIGHTS

This invention was made with government support under Contract No. DE-AC52-06 NA 25396, awarded by the U.S. Department of Energy. The governmenthas certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

More than one reissue application has been filed for U.S. Pat. No.7,638,034. The instant application Ser. No. 13/339,010, filed Dec. 28,2011 is a reissue application to U.S. Pat. No. 7,638,034 and twodivisional reissue application Ser. Nos. 14/289,445, and 14/289,388,filed May 28, 2014, and further relates to International PatentApplication No. PCT/US2012/031914, filed Apr. 2, 2012, U.S. patentapplication Ser. No. 13/437,793, filed Apr. 2, 2012, U.S. patentapplication Ser. No. 13/437,839, filed Apr. 2, 2012, U.S. patentapplication Ser. No. 13/437,817, filed Apr. 2, 2012, and U.S. patentapplication Ser. No. 13/437,753, filed Apr. 2, 2012, the entiredisclosures of which are incorporated herein by reference in theirentireties.

CROSS-REFERENCE TO RELATED APPLICATIONS

More than one reissue application has been filed for the reissue of U.S.Pat. No. 7,638,034. The reissue applications are U.S. patent applicationSer. Nos. 14/289,445, 14/289,388 and 13/339,010 (the presentapplication).

RELATED U.S. APPLICATION DATA

This application is a reissue application of U.S. Pat. No. 7,638,034,issued on Dec. 29, 2009.

BACKGROUND OF INVENTION

The invention relates to barrier structures comprising nanopores. Moreparticularly, the invention relates to structures having electroactivenanopores. Even more particularly, the invention relates toelectrochemical sensors having such structures.

The detection and identification of single molecules has receivedincreasing interest over the last few years, as there has been arealization that this can be done by analyzing transport andelectrochemical phenomena through pores having nanoscale dimensions.Measurements of the ionic current through a single-protein channelincorporated into a freestanding lipid bilayer membrane can form thebasis of a new and versatile method for single-molecule chemical andbiological sensing, called stochastic sensing. These sensors consist ofa protein pore embedded in an insulating membrane and operate bymeasuring the characteristic current through the pore in the presence ofmolecules of interest. The magnitude, duration, and rates of occurrenceof the current blockage allow rapid discrimination between similarmolecular species.

The main limitation in this nascent field is that the bulk of the workhas been focused on biologically-based stochastic sensors using proteinpores embedded in lipid bilayer membranes. The lipid bilayer membraneinto which the channel is immobilized is fragile and unstable; suchmembranes have lifetimes on the order of a few hours and, very rarely,exceed one day. These membranes are extremely delicate and susceptibleto breakage, requiring vibration isolation tables, low acoustic noiseenvironments, and special solution handling. This is unacceptable forfield-usable devices and applications outside the laboratory.Furthermore, although a range of membrane proteins, which can bemodified as desired through biochemistry or mutagenesis, may beexploited as sensors, the availability of biological pores is stilllimited with respect to having complete freedom in pore size, structure,and composition. Attempts to fabricate solid-state nanopores that areable to mimic the ion transport properties of protein ion channels lackreproducible dimensional control at the nanometer scale.

Existing biologically-based stochastic membrane sensors are notsufficiently robust for widespread use outside a controlled laboratorysetting. Therefore, what is needed is a stochastic membrane sensor thatis sufficiently robust to withstand use in applications under normalconditions. What is also needed is a membrane for a stochastic sensorthat is not biologically-based. What is further needed is a membrane fora stochastic sensor having a diameter that is reproducibly controllable.

SUMMARY OF INVENTION

The present invention meets these and other needs by providing a barrierstructure for use in an electrochemical stochastic membrane sensor forsingle molecule detection. The sensor is based upon inorganic nanoporeshaving electrically tunable dimensions. The inorganic nanopores areformed from inorganic materials and an electrically conductive polymer.Methods of making the barrier structure and sensing single moleculesusing the barrier structure are also described.

Accordingly, one aspect of the invention is to provide a barrierstructure. The barrier structure comprises: a first chamber; a secondchamber; a barrier separating the first chamber and the second chamber.The barrier comprises at least one electroactive nanopore structurejoining the first chamber and the second chamber. The at least oneelectroactive nanopore structure comprises: a wall defining aelectroactive nanopore connecting the first chamber and the secondchamber and having an electrically tunable diameter; a first electrodepair disposed in the wall, wherein electrodes of the first electrodepair are disposed at opposite ends of the electroactive nanopore, andwherein a first voltage across the first electrode pair attracts aplurality of molecules to the electroactive nanopore and drives theplurality of molecules through the electroactive nanopore; a secondelectrode pair disposed in the wall between the first electrode pair;and a conductive polymer disposed over an electrode of the secondelectrode pair, wherein the conductive polymer is responsive to a secondvoltage across the second electrode pair and is capable of expansion orcontraction in response to the second voltage, and wherein the expansiondecreases the electrically tunable diameter and the contractionincreases the electrically tunable diameter. The barrier structure alsocomprises at least one power supply electrically coupled to the firstelectrode pair and the second electrode pair, wherein the at least onepower supply provides the first voltage across the first electrode pairand the second voltage across the second electrode pair.

A second aspect of the invention is to provide an electroactive nanoporestructure. The electroactive nanopore structure comprises: a walldefining a electroactive nanopore having a first open end and a secondopen end and having a electrically tunable diameter; a first electrodepair disposed in the wall, wherein electrodes of the first electrodepair are disposed at opposite ends of the electroactive nanopore, andwherein a first voltage across the first electrode pair attracts aplurality molecules to the electroactive nanopore and drives theplurality of molecules through the electroactive nanopore; a secondelectrode pair disposed in the wall between the first electrode pair;and a conductive polymer disposed over an electrode of the secondelectrode pair, wherein the conductive polymer is responsive to a secondvoltage across the second electrode pair and is capable of expansion orcontraction in response to the second voltage, and wherein the expansiondecreases the electrically tunable diameter and the contractionincreases the electrically tunable diameter.

A third aspect of the invention is to provide a stochastic sensorstructure. The stochastic sensor structure comprising: a first chamber;a second chamber; a barrier separating the first chamber and the secondchamber, wherein the barrier comprises at least one electroactivenanopore structure joining the first chamber and the second chamber,wherein the at least one electroactive nanopore structure comprises: awall defining a electroactive nanopore connecting the first chamber andthe second chamber and having a electrically tunable diameter; a firstelectrode pair disposed in the wall, wherein electrodes of the firstelectrode pair are disposed at opposite ends of the electroactivenanopore, and wherein a first voltage across the first electrode pairattracts a plurality molecules to the electroactive nanopore and drivesthe plurality of molecules through the electroactive nanopore; and asecond electrode pair disposed in the wall between the first electrodepair; and a conductive polymer disposed over an electrode of the secondelectrode pair, wherein the conductive polymer is responsive to a secondvoltage across the second electrode pair and is capable of expansion orcontraction in response to the second voltage, and wherein the expansiondecreases the electrically tunable diameter and the contractionincreases the electrically tunable diameter; at least one power supplyelectrically coupled to the first electrode pair and the secondelectrode pair, wherein the at least one power supply provides the firstvoltage across the first electrode pair and the second voltage acrossthe second electrode pair; and a current measuring device for measuringa current flowing between the first electrode pair, wherein the currentcorresponds to a predetermined molecular species.

A fourth aspect of the invention is to provide a method of making anelectroactive nanopore structure. The electroactive nanopore structurecomprises: a wall defining a electroactive nanopore having a first openend and a second open end and having a electrically tunable diameter; afirst electrode pair having electrodes disposed at opposite ends of theelectroactive nanopore; a second electrode pair comprising a secondanode and a second cathode disposed in the wall between the firstelectrode pair; and a conductive polymer disposed over an electrode ofthe second electrode pair. The method comprises the steps of: providinga template comprising a strip of photocurable polymer; depositingalternating layers of conductive material and insulating material overthe template, wherein the alternating layers form the first electrodepair and the second electrode pair, and wherein electrodes of the firstelectrode pair and the second electrode pair are separated by at leastone layer of insulating material; removing the template to form theelectroactive nanopore; and depositing the conductive polymer on theelectrode of the second electrode pair.

A fifth aspect of the invention is to provide a method of sensing thepresence of an analyte molecule. The method comprises the steps of:providing a sensor structure, the sensor structure comprising a samplingchamber, a collection chamber, and a separation structure separating thesampling chamber and the collection chamber, wherein the separationstructure includes a electroactive nanopore structure comprising: a walldefining a electroactive nanopore connecting the sampling chamber andthe collection chamber and having a electrically tunable diameter; afirst electrode pair having electrodes disposed at opposite ends of theelectroactive nanopore; a second electrode pair disposed in the wallbetween the first electrode pair; and a conductive polymer disposed overan electrode of the second electrode pair; providing the analyte to thesampling chamber; passing the analyte molecule from the sampling chamberinto the electroactive nanopore; applying a first voltage across thefirst electrode pair; and measuring a current across the first electrodepair, wherein the current is indicative of the presence of the analytemolecule.

A sixth aspect of the invention is to provide a method of controllingflow of a fluid between a first chamber and a second chamber. The methodcomprising the steps of: providing a barrier structure, wherein thebarrier structure includes at least one electroactive nanoporestructure, wherein the at least one electroactive nanopore structurecomprises: a wall defining a electroactive nanopore connecting the firstchamber and the second chamber and having a electrically tunablediameter; a first electrode pair disposed in the wall and havingelectrodes disposed at opposite ends of the electroactive nanopore; asecond electrode pair disposed in the wall between the first electrodepair; and a conductive polymer disposed over an electrode of the secondelectrode pair; providing the fluid to the first chamber; passing thefluid from the first chamber into the electroactive nanopore; andincreasing or decreasing the electrically tunable diameter of theelectroactive nanopore to control the flow of the fluid through theelectroactive nanopore to the second chamber.

These and other aspects, advantages, and salient features of the presentinvention will become apparent from the following detailed description,the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a barrier structure;

FIG. 2 is a flow chart for a method of sensing an analyte molecule;

FIG. 3 is a flow chart for a method of making a barrier structure;

FIG. 4a is a schematic representation showing the response of theelectrically tunable diameter of an electroactive nanopore to voltageV_(Polymer);

FIG. 4b is a plot of V_(Polymer) as a function of time;

FIG. 4c is a plot of current I_(Pore) passing through the electroactivenanopore, shown in FIG. 4a, as a function of time;

FIG. 5 is a plot of V_(Polymer) and molecular diameter showingcharacteristic voltages V₁ and V₂ for molecules having diameters d₁ andd₂, respectively; and

FIG. 6 is a schematic representation of the operation of a stochasticsensor.

FIG. 7 is a flow chart for a method of controlling fluid.

DETAILED DESCRIPTION

In the following description, like reference characters designate likeor corresponding parts throughout the several views shown in thefigures. It is also understood that terms such as “top,” “bottom,”“outward,” “inward,” and the like are words of convenience and are notto be construed as limiting terms. In addition, whenever a group isdescribed as either comprising or consisting of at least one of a groupof elements and combinations thereof, it is understood that the groupmay comprise or consist of any number of those elements recited, eitherindividually or in combination with each other.

Referring to the drawings in general and to FIG. 1 in particular, itwill be understood that the illustrations are for the purpose ofdescribing a particular embodiment of the invention and are not intendedto limit the invention thereto. Turning to FIG. 1, a barrier structureof the present invention is shown. Barrier structure 100 comprises afirst chamber 110, a second chamber 120 and a barrier 130 separatingfirst chamber 110 and second chamber 120.

First chamber 110 and second chamber 120 are adapted to contain a fluid,and their dimensions and other characteristics depend on the specificapplication in which barrier structure is used. In one embodiment, forexample, first chamber 110 may serve as a sampling chamber forcollecting a fluid for analysis, and second chamber 120 may serve as ananalysis chamber. Alternatively, first chamber 110 and second chamber120 may simply be reservoirs for containing a fluid buffer, with barrier140 limiting communication between the reservoirs.

Barrier 140 comprises at least one electroactive nanopore 130 having awall defining a solid-state electroactive nanopore 130 and connectingfirst chamber 110 and second chamber 120. In one embodiment, barrier 140comprises an array of electroactive nanopores 130. A first electrodepair 134, having two electrodes disposed at opposite ends ofelectroactive nanopore 130, is disposed in the nanopore wall at oppositeends of electroactive nanopore 130. The two electrodes of firstelectrode pair 134 are proximate to where wall 142 joins first chamber110 and second chamber 120, respectively. A first voltage V_(Pore), whenapplied across first electrode pair 134, attracts a plurality ofmolecules present in either first chamber or second chamber toelectroactive nanopore 130 and drives the plurality of molecules throughelectroactive nanopore 130 and into the opposite chamber. Firstelectrode pair 134 may comprise any conductive material known in the artsuch as, but not limited to, platinum, gold, graphite, electricallyconductive metal alloys, combinations thereof, and the like.

A second electrode pair 132 comprising two electrodes is disposed inwall 142 between the electrodes of first electrode pair 134. Secondelectrode pair 132 may comprise any conductive material known in the artsuch as, but not limited to, platinum, gold, graphite, electricallyconductive metal alloys, combinations thereof, and the like.

The electrodes of first electrode pair 134 and second electrode pair 132are separated from each other by insulating material 144. Insulatingmaterial 144 comprises at least one of a metal oxide such as sapphireand silica (SiO₂), glasses, nonconductive polymers, silicon, and thelike.

A conductive polymer 136 is disposed on the surface of wall 142 over anelectrode of the second electrode pair 132. Conductive polymer 136 haselectrically tunable dimensions; i.e., it is responsive to a secondvoltage V_(Polymer), applied across second electrode pair 132, and iscapable of expanding or contracting in response to the second voltage.The presence of conductive polymer 136 on the surface of the wall ofelectroactive nanopore 130 provides the electroactive nanopore 130 withan electrically tunable diameter 138 or cross-section. As conductivepolymer 136 expands or contracts, its volume changes, causing thecross-section, or diameter 138, of electroactive nanopore 130 tocorrespondingly decrease and increase. Diameter 138 is also reversiblytunable—i.e., it may be decreased and then increased, or vice versa.Conductive polymer 136 comprises an ionically conductive polymer suchas, but not limited to, polypyrrole, polyaniline, combinations thereof,and the like.

In the absence of a second voltage V_(Polymer), electroactive nanopore130 has a diameter 138 of up to about 50 nm. With the application of thesecond voltage V_(Polymer), conductive polymer 136 may be expanded todecrease diameter 138 to a zero or near-zero value, effectively closingelectroactive nanopore 130.

The thickness of each of the electrodes 132, 134 in electroactivenanopore 130 depends on the desired electrode size and the desiredspacing between electrodes. The thicknesses of the individual layers ofinsulating material 140 must be sufficient to prevent shorting or arcingbetween the electrode layers. The individual electrodes and layers ofinsulating material 140 each have a thickness of up to 5 nm. In oneembodiment, the thickness is in a range from about 1 nm to about 5 nm.

The length of electroactive nanopore 130 should be long enough toaccommodate a single analyte molecule of interest. As analyte moleculesof interest may vary from one application to another, the length ofelectroactive nanopore 130 may be varied accordingly. Electroactivenanopore 130 may, for example, have one length when used to detect thepresence of proteins, another length when detecting polymers, and yet athird length when detecting DNA molecules. Electroactive nanopore 130may have a length in a range from about 5 nm to about 50 nm. In oneembodiment, electroactive nanopore 130 has a length of up to 5 nm, whichapproximates the length of protein pores that are used in stochasticsensors.

An example of how the current through electroactive nanopore 130 isaffected by applying second voltage V_(Polymer) across second electrodepair 132 and expanding conductive polymer 136 is illustrated in FIGS.4a, 4b, and 4c. With first voltage V_(Pore) across first electrode pair134 held constant, V_(Polymer) is increased from a low value (A in FIG.4b) to a medium value (B) to a high value (C). Conductive polymer 136correspondingly expands (FIG. 4a), narrowing the diameter of theelectroactive nanopore. As diameter 138 decreases (and conductivepolymer expands), the current through electroactive nanopore 130decreases as well (FIG. 4c).

A characteristic voltage corresponding to the second voltage (whilemaintaining first voltage V_(Pore) across first electrode pair 134 at aconstant value) may be applied across second electrode pair 132 to tunediameter 138 to the approximate size. The application of characteristicvoltages V₁ and V₂ for molecules having sizes of d₁ and d₂,respectively, is shown in FIG. 5. By applying voltage V₁ across secondelectrode pair 132, diameter 138 is tuned to the size of an analytemolecule having diameter d₁ while effectively preventing largermolecules having diameter d₂ from passing through electroactive nanopore130.

At least one power source (not shown) is electrically coupled to firstelectrode pair 134 and second electrode pair 132, and provides the firstvoltage across first electrode pair 134 and second voltage across secondelectrode pair 132. The power source may be either a DC power source oran AC power source.

In one embodiment, barrier structure 100 forms a portion of asingle-molecule—or stochastic—sensor that is adapted to detectparticular species of analyte molecules present in a fluid. Such asensor operates by measuring a characteristic current throughelectroactive nanopore 130 in the presence of analyte molecules ofinterest. The magnitude, duration, and rates of occurrence of thecurrent blockage by the analyte molecule allow rapid discriminationbetween similar molecular species. Whereas previous stochastic sensorsformed using protein pores embedded in lipid membranes are fragile andunstable, barrier structure 100 and electroactive nanopore 130 arestructurally stable, due to their construction from inorganic materialsand conductive polymers, and are capable of repeated use.

The selectivity of the stochastic sensor is based on the characteristiccurrents associated with the flow of different types of molecules in anionic aqueous solution. The molecules have multiple measurableparameters that allow discrimination between different—butsimilar—species. For a selected diameter 138, each type of analytemolecule exhibits a different characteristic current and noisesignature.

The stochastic sensor includes two buffer reservoirs, which areanalogous to first chamber 110 and second chamber 120, joined by atleast one electroactive nanopore 130. To detect the analyte molecule,first voltage V_(Pore) is applied across first electrode pair 134,driving molecules through electroactive nanopore 130. The voltageapplied between the second electrode pair 132 causes conductive polymer136 to either expand or contract, thus controlling the diameter 138 ofelectroactive nanopore 130.

The selectivity of the stochastic sensor is based on the characteristiccurrent signature associated with the flow of each type of analytemolecule through electroactive nanopore 130. Small ions flow throughelectroactive nanopore 130, producing a current having a relatively highvalue. When an analyte molecule having a diameter that is less thandiameter 138 of electroactive nanopore 130 passes through the nanopore,the molecule partially occludes the passage of ions, thereby causing thecurrent to decrease. After the analyte molecule traverses electroactivenanopore 130, normal ion flow through the nanopore resumes and thecurrent is restored to its initial value. If an analyte molecule that islarger than diameter 138 of electroactive nanopore 130 tries to traversethe nanopore, the passage of ions through the nanopore is blocked andthe current drops to zero. The polarity of first electrode pair 134 mustthen be reversed to unblock the nanopore. When the inner diameter 138 ofelectroactive nanopore 130 is increased by changing V_(Polymer) to allowthe analyte molecule to pass through the nanopore, the size of themolecule and its electrodynamic interactions with the charges inconductive polymer 136 will determine the current drop that is observedas the analyte molecule traverses electroactive nanopore 130.

Electroactive nanopore 130 can be electrochemically characterized byperforming cyclic voltammetry between the electrodes of second electrodepair 132 in the presence of a buffer while maintaining a constantvoltage across first electrode pair 134. The electrochemical behavior ofelectroactive nanopore 130 can then be characterized using the recordedcyclic voltammograms and the current across electroactive nanopore 130.Electroactive nanopore 130 is then closed by applying the appropriatevoltage V_(Polymer) across second electrode pair 132 while applying aconstant independent voltage V_(Pore) across first electrode pair 134and monitoring the current through the nanopore. This yields a referencecurrent for a state where substantially no molecules or ions—or aminimum number of molecules or ions—are passing through electroactivenanopore 130. Next, a sample containing a first analyte molecularspecies is introduced into either first chamber 110 or second chamber120, and voltage V_(Polymer) across second electrode pair 132 isdecreased to slowly contract conductive polymer 136 and openelectroactive nanopore 130. The resulting increase in diameter 138 ofthe nanopore results in a corresponding increase in ionic currentthrough the nanopore. The characteristic voltage V_(Polymer) associatedwith the first analyte molecular species is the voltage associated withthe passage of the first analyte species through the nanopore.

FIG. 6 illustrates the principle of operation of the stochastic sensor.Initially, only small ions flow through electroactive nanopore 130,procuring a current having a relatively high value ((a) in FIG. 6). Asone type of analyte molecule 160 that is smaller than diameter 138passes through electroactive nanopore 130, the analyte molecule 160partially occludes the passage of ions, causing the current to drop ((b)in FIG. 6). After the molecule has traversed electroactive nanopore 130,the current is restored to its original value, as shown in (b). In (c),a second type of analyte molecule 162, larger than diameter 138, triesto traverse electroactive nanopore 130. The passage of ions throughelectroactive nanopore 130 is completely blocked and the current goes tozero. Here, electroactive nanopore 130 may be unblocked by reversing thefirst voltage V_(Pore). In (d), diameter 138 is increased by changingV_(Polymer). The flow of ions—and the current—through electroactivenanopore 130 then resumes. The size of the second analyte molecule 162and its electrodynamic interactions with the charges in conductivepolymer 136 will determine the current drop when the molecule traverseselectroactive nanopore 130. Once the second analyte molecule 162 exitselectroactive nanopore 130, the current returns to its original value,as shown in (d).

The stochastic currents associated with molecules of the same speciespassing through electroactive nanopore 130 are monitored for laterstatistical analysis, which provides parameters, such as blockagecurrents, blockage times, blockage frequencies, current distribution,signal-to-noise ratios, and the like, that are used together with thecharacteristic voltage for identification of the analyte molecule.

If the analyte sample includes a mixture of molecules, random drops incurrent to either positive values or the reference current may occur, assome molecules pass through the electroactive nanopore 130 while othersblock the entrance to the nanopore. In such cases, the characterizationprocess is typically repeated, and the voltage V_(Polymer) across secondelectrode pair 132 is adjusted to the characteristic voltage of eachanalyte molecular species. In addition, the stochastic current ismonitored for analytical purposes.

The stochastic sensor described hereinabove incorporates for the firsttime two important transport-selectivity capabilities into the field ofsensor development. First, because diameter 138 of electroactivenanopore 130 can be modified in a controllable manner, the sensor can beused to cleanly separate different molecules on the basis of molecularsize, ranging from simple ions to complex compounds and evenmicroorganisms. Second, because the conductive polymer 136 can becharged in an ionic solution, the stochastic sensor can discriminatebetween molecules of similar size based on their differentelectrodynamic interactions with the conducting polymer.

Furthermore, the use of solid-state electroactive nanopores such asthose described herein provides a significant advantage, as fabricationof an array of several pores can be integrated with electronics andon-chip computational hardware to provide a portable device capable ofperforming multiple sensing functions. Unlike sensors based onbiological membranes and protein channels, this robust sensor will bestable and functional over a wider range of temperatures, solvents,voltages, and other potentially adverse conditions.

This new technology not only could be used in sensing but also inanalytical chemistry, specifically in bio-separations, electroanalyticalchemistry, and in the development of new approaches to DNA sequencingbased on transport through the electroactive nanopore.

In another embodiment, barrier structure 100 forms a portion of a valvestructure. Here, conductive polymer 136 may expand or contract inresponse to changes in voltage V_(Polymer) across second electrode pair132. As conductive polymer 136 expands or contracts, the tunablediameter 138 of electroactive nanopore 130 either decreases orincreases, thereby regulating flow between first chamber 110 and secondchamber 120.

In yet another embodiment, barrier structure 100 is a membrane thatseparates first chamber 110 and second chamber 120. Here, barrierstructure 100 includes an array of electroactive nanopores 130. Based onthe characteristic voltage signature associated with the flow ofdifferent types of molecules through electroactive nanopore 130, themembrane may be selectively tuned to allow certain molecular species topass from first chamber 110 to second chamber 120.

The invention also includes a method of sensing an analyte molecule. Aflow chart outlining the method is shown FIG. 2. In Step 210, a sensorstructure comprising a sampling chamber, a collection chamber, and aseparation structure is provided. The separation structure includes atleast one electroactive nanopore 130, described herein and shown inFIG. 1. An analyte molecule in a buffer solution is provided to thesampling chamber (Step 220). The analyte molecule then passes from thesampling chamber into the electroactive nanopore in Step 230 by applyinga first voltage V_(Pore) across first electrode pair 134. As previouslydescribed herein, a current across first electrode pair 134 is generatedby ions in the buffer solution passing through electroactive nanopore130. When an analyte molecule having a diameter that is less than thediameter of electroactive nanopore 130 passes through the nanopore, themolecule partially occludes the passage of ions, thereby causing thecurrent to decrease. After the analyte molecule traverses electroactivenanopore 130, normal ion flow through the nanopore resumes and thecurrent is restored to its initial value. The size of the analytemolecule and its electrodynamic interactions with the charges inconductive polymer 136 will determine the current drop that is observedas the analyte molecule traverses electroactive nanopore 130.Accordingly, the current across the first electrode pair 132 is measuredin Step 240 to determine whether the analyte molecule is present.

The invention also provides a method of making barrier structure 100having electroactive nanopore 130. A flow chart of method 300 is shownin FIG. 3. In Step 310, a template is provided. The template comprises astrip of a photocurable polymer such as a polyimide or the like. Thepolymer strip, which is typically a few centimeters in length and lessthan 1 mm wide, is deposited on an insulating material such as sapphire,glass, or a silicon wafer. In one embodiment, the template includes apolymeric cylinder comprising the same photocurable polymer. Thepolymeric cylinder is vertically placed on top of the polymer strip. Thepolymeric cylinder has a diameter that is substantially equal to thedesired maximum diameter of the electroactive nanopore. In oneembodiment, the polymeric cylinder has a diameter of about 50 nm and aheight of about 200 nm. In another embodiment, the polymeric cylinder isnot provided.

In Step 320, alternating layers of conductive material and insulatingmaterial are deposited over the template to form a first—orouter—electrode pair and a second—or inner—electrode pair separated byat least one layer of insulating material. In one embodiment, theconductive material may comprise any conductive material known in theart such as, but not limited to, platinum, gold, graphite, conductivemetal alloys known in the art, combinations thereof, and the like. Theinsulating material comprises at least one of a metal oxide, such assapphire or silica (SiO₂), glasses, nonconductive polymers, silicon, orthe like.

The thicknesses of the individual layers of insulating material must besufficient to prevent shorting or arcing between the electrode layers.The thickness of the individual layers of conductive material depends onthe desired electrode size and distance between electrodes. Theindividual layers of conductive and insulating material each have athickness of up to 5 nm. In one embodiment, the thickness is in a rangefrom about 1 nm to about 5 nm.

In one embodiment, the conductive layers and insulating layers aredeposited using energetic neutral atom beam lithography/epitaxy (alsoreferred to herein as “ENABLE”), which is described in U.S. ProvisionalPatent Application 60/738,624, filed on Nov. 21, 2005, by Mark A.Hoffbauer et al., entitled “Method of Forming Nanostructures on aSubstrate,” the contents of which are incorporated herein in theirentirety.

The template is then removed (Step 330), typically by dissolution of thephotocurable polymer. Where a polymer cylinder is provided, dissolutionof the template leaves a microfluidic channel—or chamber—having ananopore on top. In embodiments in which the template does not includethe polymeric cylinder described above, the nanopore may be formed bydrilling through the deposited conductive and insulating layers using afocused ion beam. The nanopore diameter reflects the size of thepolymeric cylinder used in the template, and is typically about 50 nm. Asecond microfluidic channel or chamber is then formed from a polymericmaterial such as polydimethyl siloxane or the like. The secondmicrofluidic chamber is then placed on top of the first microfluidicchamber such that the electroactive nanopore is enclosed between—andconnects—the two chambers.

In Step 340, a conductive polymer film is electrochemically deposited onone electrode of the second electrode pair. The thickness of theconductive polymer film that is actually deposited depends on thediameter of the nanopore, in one embodiment, the thickness of theconductive polymer film is in a range from about 10 nm to about 50 nm.The conductive polymer comprises an ionic conductive polymer such as,but not limited to, polypyrrole, polyaniline, combinations thereof, andthe like.

Method 300 can be optimized and updated for later fabrication of anarray of several electroactive nanopores. The nanopores can beintegrated with electronics and on-chip computational hardware to domultiple sensing in a portable device.

The invention also provides a method of controlling fluid from a firstchamber to a second chamber. A flow chart for method 700 is shown inFIG. 7. in step 710, a barrier structure, such as barrier structure 100including at least one electro active nanopore 130 describedhereinabove, is provided. Fluid is provided to the first chamber (Step720) and is passed into the electroactive nanopore (Step 730). Step 730is accomplished by applying a first voltage across first electrode pair134 in electroactive nanopore 130. The first voltage is sufficient tocause the fluid to migrate from the first chamber through electroactivenanopore 130 to the second chamber. In Step 740, the electricallytunable diameter 138 of electroactive nanopore is either increased ordecreased to control the flow of the fluid through electroactivenanopore 130 to the second chamber. The electrically tunable diametermay be either decreased or increased by applying a second voltage acrosssecond electrode pair 132 of electrode active nanopore 130. The secondelectrode voltage causes conductive polymer 136 to either expand orcontract, which correspondingly causes electrically tunable diameter 138to either decrease or increase.

While typical embodiments have been set forth for the purpose ofillustration, the foregoing description should not be deemed to be alimitation on the scope of the invention. Accordingly, variousmodifications, adaptations, and alternatives may occur to one skilled inthe art without departing from the spirit and scope of the presentinvention.

The invention claimed is:
 1. A barrier structure, the barrier structurecomprising: a. a first chamber; b. a second chamber; c. a barrierseparating the first chamber and the second chamber, wherein the barriercomprises at least one electroactive nanopore structure joining thefirst chamber and the second chamber, wherein the at least oneelectroactive nanopore structure comprises: i. a wall defining aelectroactive nanopore connecting the first chamber and the secondchamber and having an electrically tunable diameter; ii. a firstelectrode pair disposed in the wall, wherein electrodes of the firstelectrode pair are disposed at opposite ends of the electroactivenanopore, and wherein a first voltage across the first electrode pairattracts a plurality molecules to the electroactive nanopore and drivesthe plurality of molecules through the electroactive nanopore; and iii.a second electrode pair disposed in the wall between the first electrodepair; and iv. a conductive polymer disposed over an electrode of thesecond electrode pair, wherein the conductive polymer is responsive to asecond voltage across the second electrode pair and is capable ofexpansion or contraction in response to the second voltage, and whereinthe expansion decreases the electrically tunable diameter and thecontraction increases the electrically tunable diameter; and d. at leastone power supply electrically coupled to the first electrode pair andthe second electrode pair, wherein the at least one power supplyprovides the first voltage across the first electrode pair and thesecond voltage across the second electrode pair.
 2. The barrierstructure according to claim 1, further including a current measuringdevice for measuring a current flowing between the first electrode pair,wherein the current corresponds to a predetermined molecular species. 3.The barrier structure according to claim 2, wherein the barrierstructure forms a portion of a sensor.
 4. The barrier structureaccording to claim 1, wherein each of the electrodes of the firstelectrode pair and the second electrode pair comprises one of platinum,gold, graphite, a metal alloy, and combinations thereof.
 5. The barrierstructure according to claim 1, wherein the first electrode pair and thesecond electrode pair are separated by an insulating material.
 6. Thebarrier structure according to claim 1, wherein the insulating materialcomprises at least one of a glass, a metal oxide, a non-conductivepolymer, and combinations thereof.
 7. The barrier structure according toclaim 1, wherein the conductive polymer is one of polypyrrole,polyaniline, and combinations thereof.
 8. The barrier structureaccording to claim 1, wherein the barrier structure forms a portion ofone of a valve structure and a membrane structure.
 9. The barrierstructure according to claim 1, wherein the at least one power supplyincludes a DC power supply.
 10. An electroactive nanopore structure, theelectroactive nanopore structure comprising: a. a wall defining aelectroactive nanopore having a first open end and a second open end andhaving a electrically tunable diameter; b. a first electrode pairdisposed in the wall, wherein electrodes of the first electrode pair aredisposed at opposite ends of the electroactive nanopore, and wherein afirst voltage across the first electrode pair attracts a pluralitymolecules to the electroactive nanopore and drives the plurality ofmolecules through the electroactive nanopore; and c. a second electrodepair disposed in the wall between the first electrode pair; and d. aconductive polymer disposed over an electrode of the second electrodepair, wherein the conductive polymer is responsive to a second voltageacross the second electrode pair and is capable of expansion orcontraction in response to the second voltage, and wherein the expansiondecreases the electrically tunable diameter and the contractionincreases the electrically tunable diameter.
 11. The electroactivenanopore according to claim 10, wherein each of the electrodes of thefirst electrode pair and the second electrode pair comprises one ofplatinum, gold, graphite, a metal alloy, and combinations thereof. 12.The electroactive nanopore structure according to claim 10, wherein thefirst electrode pair and the second electrode pair are separated by aninsulating material.
 13. The electroactive nanopore structure accordingto claim 10, wherein the insulating material comprises a glass, a metaloxide, a non-conductive polymer, and combinations thereof.
 14. Theelectroactive nanopore structure according to claim 10, wherein theconductive polymer is one of polypyrrole, polyaniline, and combinationsthereof.
 15. The electroactive nanopore structure according to claim 10,wherein the electroactive nanopore structure forms a portion of one of avalve structure, a sensor, and a membrane structure.
 16. A stochasticsensor structure, the stochastic sensor structure comprising: a. a firstchamber; b. a second chamber; c. a barrier separating the first chamberand the second chamber, wherein the barrier comprises at least oneelectroactive nanopore structure joining the first chamber and thesecond chamber, wherein the at least one electroactive nanoporestructure comprises: i. a wall defining a electroactive nanoporeconnecting the first chamber and the second chamber and having aelectrically tunable diameter; ii. a first electrode pair disposed inthe wall, wherein electrodes of the first electrode pair are disposed atopposite ends of the electroactive nanopore, and wherein a first voltageacross the first electrode pair attracts a plurality molecules to theelectroactive nanopore and drives the plurality of molecules through theelectroactive nanopore; and iii. a second electrode pair disposed in thewall between the first electrode pair; and iv. a conductive polymerdisposed over an electrode of the second electrode pair, wherein theconductive polymer is responsive to a second voltage across the secondelectrode pair and is capable of expansion or contraction in response tothe second voltage, and wherein the expansion decreases the electricallytunable diameter and the contraction increases the electrically tunablediameter; d. at least one power supply electrically coupled to the firstelectrode pair and the second electrode pair, wherein the at least onepower supply provides the first voltage across the first electrode pairand the second voltage across the second electrode pair; and e. acurrent measuring device for measuring a current flowing between thefirst electrode pair, wherein the current corresponds to a predeterminedmolecular species.
 17. A method of making a electroactive nanoporestructure, wherein the electroactive nanopore structure comprises: awall defining a electroactive nanopore having a first open end and asecond open end and having a electrically tunable diameter; a firstelectrode pair having electrodes disposed at opposite ends of theelectroactive nanopore; a second electrode pair comprising a secondanode and a second cathode disposed in the wall between the firstelectrode pair; and a conductive polymer disposed over an electrode ofthe second electrode pair; the method comprising the steps of: a.providing a template comprising a strip of photocurable polymer; b.depositing alternating layers of conductive material and insulatingmaterial over the template, wherein the alternating layers form thefirst electrode pair and the second electrode pair, and whereinelectrodes of the first electrode pair and the second electrode pair areseparated by at least one layer of insulating material; c. removing thetemplate to form the electroactive nanopore; and d. depositing theconductive polymer on the electrode of the second electrode pair to formthe electrically tunable diameter.
 18. The method according to claim 17,wherein the step of depositing alternating layers of conductive materialand insulating material over the template comprises: a. depositing afirst conductive layer over the template; b. depositing a firstinsulating layer over the first conductive layer; c. depositing a secondconductive layer over the first insulating layer; d. depositing a secondinsulating layer over the second conductive layer; e. depositing a thirdconductive layer over the second conductive layer, wherein the secondconductive layer and the third conductive layer form the secondelectrode pair; f. depositing a third insulating layer over the thirdconductive layer; and g. depositing a fourth conductive layer over thethird conductive layer, wherein the first conductive layer and thefourth conductive layer form the first electrode pair.
 19. The methodaccording to claim 17, wherein at least one of the first conductivelayer, the first insulating layer, the second conductive layer, thesecond insulating layer, the third conductive layer, the thirdinsulating layer, and the fourth conductive layer is deposited byenergetic neutral beam lithography/epitaxy.
 20. The method according toclaim 17, wherein the step of depositing the conductive polymercomprises electrochemically depositing the conductive polymer onto atleast one of the second anode and the second cathode.
 21. The methodaccording to claim 17, wherein the template further comprises a cylinderhaving a diameter that is substantially equal to the electricallytunable diameter of the electroactive nanopore, wherein the cylindercomprises the photcurable polymer.
 22. The method according to claim 17,wherein the step of removing the template to form the electroactivenanopore comprises drilling through the alternating layers of conductivematerial and insulating material with a focused ion beam to form theelectroactive nanopore.
 23. A method of sensing the presence of ananalyte molecule, the method comprising the steps of: a. providing asensor structure, the sensor structure comprising a sampling chamber, acollection chamber, and a separation structure separating the samplingchamber and the collection chamber, wherein the separation structureincludes a electroactive nanopore structure comprising: a wall defininga electroactive nanopore connecting the sampling chamber and thecollection chamber and having a electrically tunable diameter; a firstelectrode pair having electrodes disposed at opposite ends of theelectroactive nanopore; a second electrode pair disposed in the wallbetween the first electrode pair; and a conductive polymer disposed overan electrode of the second electrode pair; b. providing the analytemolecule to the sampling chamber; c. passing the analyte molecule fromthe sampling chamber into the electroactive nanopore; and d. measuring acurrent across the first electrode pair, wherein the current isindicative of the presence of the analyte molecule.
 24. The methodaccording to claim 23, wherein the step of passing the analyte from thesampling chamber into the electroactive nanopore comprises applying afirst voltage across the first electrode pair, wherein the first voltageis sufficient to cause the analyte to migrate from the sampling chamberthrough the electroactive nanopore structure to the collection chamber.25. The method according to claim 23, further including the step ofincreasing or decreasing the electrically tunable diameter of theelectroactive nanopore.
 26. The method according to claim 23, whereinthe step of increasing or decreasing the electrically tunable diameterof the electroactive nanopore comprises applying a second voltage acrossthe second electrode pair, wherein the second electrode voltage causesthe conductive polymer to either expand or contract.
 27. A method ofcontrolling flow of a fluid between a first chamber and a secondchamber, the method comprising: a. providing a barrier structure,wherein the barrier structure includes at least one electroactivenanopore structure, wherein the at least one electroactive nanoporestructure comprises: a wall defining a electroactive nanopore connectingthe first chamber and the second chamber and having a electricallytunable diameter; a first electrode pair disposed in the wall and havingelectrodes disposed at opposite ends of the electroactive nanopore; asecond electrode pair disposed in the wall between the first electrodepair; and a conductive polymer disposed over an electrode of the secondelectrode pair; b. providing the fluid to the first chamber; c. passingthe fluid from the first chamber into the electroactive nanopore; and d.increasing or decreasing the electrically tunable diameter of theelectroactive nanopore to control the flow of the fluid through theelectroactive nanopore to the second chamber.
 28. The method accordingto claim 27, wherein passing the fluid from the first chamber into theelectroactive nanopore comprises applying a first voltage across thefirst electrode pair, wherein the first voltage is sufficient to causethe fluid to migrate from the first chamber through the electroactivenanopore structure to the second chamber.
 29. The method according toclaim 27, wherein the step of increasing or decreasing the electricallytunable diameter of the electroactive nanopore comprises applying asecond voltage across the second electrode pair, wherein the secondelectrode voltage causes the conductive polymer to either expand orcontract, and wherein expansion of the conductive polymer increases theelectrically tunable diameter and contraction of the conductive polymerdecreases the electrically tunable diameter.
 30. A nanopore structurecomprising a nanopore having an opening and a wall defining thenanopore, wherein the opening has an electrically tunable diameter. 31.A barrier structure comprising: a first chamber; a second chamber; abarrier separating the first chamber and the second chamber, wherein thebarrier comprises at least one nanopore structure of claim 30 joiningthe first chamber and the second chamber.
 32. The nanopore structure ofclaim 30, further comprising a first electrode pair, a second electrodepair between the first electrode pair, a conductive polymer disposedover an electrode of the second electrode pair, wherein the opening isan opening through the polymer.
 33. The nanopore structure of claim 32,wherein the polymer is a conductive polymer.
 34. A nanopore structurecomprising a nanopore having an opening and a wall defining thenanopore, and one or more polymers on the wall, wherein the opening iselectrically tunable.
 35. A barrier structure comprising: a firstchamber; a second chamber; a barrier separating the first chamber andthe second chamber, wherein the barrier comprises at least one nanoporestructure of claim 34 joining the first chamber and the second chamber.36. The nanopore structure of claim 34, wherein the one or more polymerscomprise a conductive polymer.
 37. The nanopore structure of claim 34,further comprising a first electrode.
 38. The nanopore structure ofclaim 37; wherein at least part of the one or more polymers is disposedover the first electrode.
 39. The nanopore structure of claim 37,further comprising a second electrode and a third electrode, the firstelectrode being between the second electrode and the third electrode.40. A method of controlling flow of a fluid through the nanoporestructure of claim 30, the method comprising: passing the fluid throughthe opening; and tuning the electrically tunable diameter of theopening, so as to control flow of the fluid through the nanopore.
 41. Amethod of controlling flow of an ionic species through the nanoporestructure of claim 30, the method comprising: attracting the ionicspecies by a voltage through the opening; and tuning the electricallytunable diameter of the opening, so as to control flow of the ionicspecies through the nanopore.
 42. A method of making a nanoporestructure comprising a nanopore having an opening, and a wall definingthe nanopore, the method comprising electrochemically depositing one ormore materials on the wall, and forming the nanopore structure, whereinthe opening is electrically tunable.
 43. The method of claim 42, whereinthe one or more materials comprise one or more polymers.
 44. The methodof claim 42, wherein the one or more materials comprise one or moreelectrical conductors.
 45. A method of using the nanopore structure ofclaim 30, the method comprising passing an analyte molecule into thenanopore; and measuring a current through the nanopore.
 46. A method ofusing the nanopore structure of claim 34, the method comprising passingan analyte molecule into the nanopore; and measuring a current throughthe nanopore.
 47. A method of making a nanopore structure, the methodcomprising depositing a conductive layer over a template, the templatecomprising a cylinder; depositing an insulating layer over theconductive layer; removing the template to leave a nanopore through theconductive layer and the insulating layer; and depositing a conductivepolymer, wherein the template comprise a strip of photocurable polymer.48. The method of claim 47, wherein the cylinder comprises aphotocurable polymer.
 49. The method of claim 47, wherein the insulatinglayer comprises at least one of a metal oxide, glasses, nonconductivepolymers, and silicon.
 50. The method of claim 47, wherein theconductive layer comprises platinum, gold, graphite, conductive metalalloys, or a combination thereof.
 51. A method of making a nanoporestructure, the method comprising depositing a conductive layer over atemplate; depositing an insulating layer over the conductive layer;removing the template; forming a nanopore through the conductive layerand the insulating layer by a focused ion beam to expose the conductivelayer in the nanopore; and depositing a conductive polymer, wherein thetemplate comprise a strip of photocurable polymer.
 52. The method ofclaim 51, wherein the insulating layer comprises at least one of a metaloxide, glasses, nonconductive polymers, and silicon.
 53. The method ofclaim 51, wherein the conductive layer comprises platinum, gold,graphite, conductive metal alloys, or a combination thereof.
 54. Adevice comprising the nanopore structure of claim
 30. 55. A devicecomprising the nanopore structure of claim
 31. 56. A device comprisingthe barrier structure of claim
 34. 57. A device comprising the barrierstructure of claim
 35. 58. A sensor comprising the nanopore structure ofclaim
 30. 59. A sensor comprising the nanopore structure of claim 31.60. A sensor comprising the barrier structure of claim
 34. 61. A sensorcomprising the barrier structure of claim 35.